Throughout this application, various publications are referenced to provide background information concerning the state of the art as known to those of ordinary skill therein as of the date of the invention disclosed and claimed herein. In the text of this application, these publications will be referred to by full citations. The disclosures of these references in their entireties are hereby incorporated by reference into the present application.
Since the discovery of an oncorna virus--the mouse mammary tumor virus (MMTV)--capable of initiating mammary tumors in mice, efforts were made to find a similar agent in the human disease. A great body of evidence was accumulated in the last decade suggesting that human breast tumors are similar to the mouse mammary tumors. The similarities mentioned in the literature refer to particles isolated from human tumors with biochemical and biophysical properties of oncorna viruses. Furthermore, it was demonstrated that human breast tumors contain RNA molecules with detectable amounts of sequence homology to the MMTV.
More direct immunological cross-reactivity between the human tumors and the MMTV was demonstrated by Mesa-Tejada et al. 1978, J. of Histochem. and Cytochem. 26: 532-541. Using the immunoperoxidase technique, they showed that some human breast tumors (212 out of 447 patients) contain an antigen that cross-reacts with the envelope glycoprotein of mouse mammary tumor virus (glycoprotein 52000 dalton MW--gp52). Furthermore, that group was able to demonstrate that the immunological cross-reactivity is due to the protein moiety of this molecule and not to the carbohydrate fraction. These findings were very encouraging in view of the findings obtained in the mouse disease. Spiegelman's group, Ritzi et al., 1976, Virology 75: 188 and Ritzi et al., 1977, J. Exp. Med. 145: 988-1013, have demonstrated that the gp52 viral glycoprotein is an excellent indicator for the mouse disease status. Plasma levels of gp52 measured by radioimmune assay could be correlated with the existence, size and recurrence of the mouse mammary tumor after surgical excision, often without physical sign of the disease.
More recently, Dion et al., 1974, J. Virology 14: 40-46, isolated a human milk protein that is structurally and antigenically related to MMTV gp52. Immunoprecipitation analyses indicated the presence of an antigenically cross-reacting glycoprotein having a MW of about 58,000 daltons. The two glycoproteins shared common sequences when tryptic peptide maps were prepared, but they also differed significantly so that it was clear that the two glycoproteins were not identical.
Further support linking breast cancer and MMTV antigens comes from the finding that the presence of antibodies to murine mammory tumor viral antigen have been reported in the serum of a number of breast cancer patients by Witkin et al., 1980, Int. J. Cancer 25, 721-725.
While this research has important implications for the understanding of the etiology of breast cancer, it has also been of interest because of its potential for diagnosing and monitoring the cause of malignant breast disease.
Spiegelman (U.S. Pat. No. 4,379,839) discloses an immunologic method for assaying the presence of viral related proteins in plasma samples as a way of diagnosing and following breast cancer in humans. Spiegelman's assay utilizes the cross-reactivity of the viral related protein with antibodies directed to Mason-Pfizer Monkey Virus or murine mammary tumor virus. However Spiegelman's test, which does not employ monoclonal antibodies, does not give a very high percentage of clear positive reactions with all breast adenocarcinoma samples.
More recently, Mesa-Tejada et al., Breast Cancer Research Conference, Denver, Colo. Mar. 20-24, 1983 Immunology Poster Abstracts 96-97, reported studies of monoclonal antibodies to RNA virus-like from the T47D breast carcinoma cell line. They reported on ascites form of two monoclonal antibodies (ASC.sub.2 18, ASC.sub.2 26), to virus-like particles isolated from the T47D clone 11 cells, the first giving 91% positive staining, the latter giving 58% positive staining. However, ASC.sub.2 18 gave 63% positive staining in the samples of benign breast tissue tested while ASC.sub.2 46 gave 26% positive staining in the samples of benign breast tissue tested. However, such a high rate of false positives, in immunohistochemical assay, is unsatisfactory for diagnosis of breast carcinomas.
Two other research groups have tried to produce McAbs reacting specifically with human mammary tumor-Ag. Papadimitriou et al., 1981, Int. J. Cancer 28: 17-21, isolated three hybridomas producing McAbs against components of the human mammary fat globules. A11 three Abs showed negative reactions with fibroblasts, lymphoblastoid cells, and a large number of epithelial cell lines of non-breast origin. Eight breast cancer lines were tested and the results obtained were that the two of the Abs reacted with seven breast cell lines, and the third Ab reacted with only two out of the eight breast cell lines. All three McAbs isolated by Papadimitriou bound positively to a pharingeal carcinoma line and a colon carcinoma line; supernatants from two of the three McAbs showed binding to derivatives of HeLa cells (Papadimitriou et al., 1981, Int. J. Cancer 28: 17-21).
Schlom et al., 77: 6841-6845 (1980, Proc. Nat'l, Acad. Sci. (USA) 78: 3199-3203, fused lymphocytes from lymph nodes obtained at mastectomy from breast cancer patients with myeloma cells NS-1 (Balb/c non Ig-secreting myeloma cell line), and obtained hybridoma cultures that synthesized human monoclonal Ab. The immunological reactivities of the human Igs were assayed on tissue sections by using the immunoperoxidase technique. The human Ig M McAb.MBE6, was chosen for extensive anslysis because of its reactivity against human breast carcinomas, and the results obtained were variable: (1) Eighty-one percent of primary malignant mammary tumors and 100% of metastatic breast lesions reacted positively with moderate or strong intensity; (2) fourteen percent of benign breast lesions showed staining; (3) moreover, normal mammary epithelial cells in areas adjacent to primary tumor cells showed staining with the MBE6; (4) preliminary studies also indicated a cross-reactivity between the MBE6 and cells of selected non-breast adenocarcinomas, such as bronchio-alveolar carcinoma of the lung and a medullary carcinoma of the thyroid. As mentioned by Schlom et al., this McAb is not entirely specific to mammary breast tumors since it reacts with a small population of normal breast tissue and other malignant tissues.
Although improvements in immunoassays for human breast cancer have provided increased sensitivity or specificity, or both, it has not been possible until the present invention to obtain a high positive staining rate without having an associated high rate of false positives.